<P><i><b> Structure and Function of Glycosyltransferases: </i></b> Structural studies on glycosyltransferases have revealed that: (I) The glycosyltransferases have flexible loop(s) which undergo conformational changes upon donor substrate binding and create the acceptor binding site, and binding site for add-on domains. (II) A few residues in the catalytic pocket determine the donor sugar specificity of the enzyme. (III) In the metal-ion dependent glycosyltransferases, the binding site for the metal ion is located at the N-terminal hinge region of the flexible loop. </p><P><i><b> Flexible loops in the glycosyltransferases upon donor substrate binding undergo conformational changes and interact with add-on domains: </i></b> To date, the detailed structure-function studies on glycosyltransferases, in particular on beta1,4-galactosyltransferase-1 (b4Gal-T1) from our laboratory, have shown that in the vicinity of their catalytic pocket are one or two flexible loops that, upon binding of the nucleotide sugar donor substrate and the metal ion (when required as a cofactor), change the conformation often from open to closed, creating an acceptor binding site on the enzyme that did not exist before. After the transfer of the glycosyl unit to the acceptor, the product is ejected, and the loop reverts to its native conformation to release the remaining nucleotide moiety. In the metal-ion dependent enzymes, the metal ion binding site is generally at the amino terminal hinge region of the flexible loop. </p> <p> To diversify the catalytic activity towards less preferred substrates, such as sugar acceptors or proteins or lipids or aglycons, the catalytic domains of glycosyltransferases either interact (1) with an additional protein, or have acquired add-on domains at the C-terminus or acquired add-on domains at the N-terminus. For example, in the lactose synthase enzyme, the b4Gal-T1, after conformational changes in the flexible loops to a closed conformation, interacts with a mammary gland-specific protein, alpha-lactalbumin (LA) at its carboxyl terminal end, changing the acceptor specificity of the enzyme towards less preferred acceptor glucose. LA protein, although not linked to b4Gal-T1, acts as an add-on domain. Several other glycosyltransferases have been shown or suggested to require an activating protein which may interact in a manner similar to the lactose synthase system, but no structural studies are as yet available on these cases. In contrast to two interacting proteins, the catalytic domains of polypeptide a-N-Acetylgalactosaminyltransferases (ppGalNAc-Ts) have a lectin domain that is linked to at the C-terminus of the catalytic domain via a linker region and determines the specificity towards a peptide or a glycopeptide. The loops in the catalytic domain of these enzymes also undergo a conformational change upon binding of the metal ion and the sugar donor, while the lectin domain moves, bringing in the bound glycopeptide acceptor in the catalytic pocket, in order to synthesize O-a-GalNAc moiety on the glycopeptide. Also in this category is the alpha-1,6-Fucosyltransferase (FUT8), where an SH3 domain has been identified that is linked at the C-terminus of the catalytic domain.</p><P><i><b> A few residues in the catalytic pocket determine the donor sugar specificity of glycosyltransferases: Role of a single amino acid in the evolutionary divergence of invertebrate and vertebrate glycoconjugates:</i></b> Based on the structural information, we have previously shown, that the residue Tyr/Phe289 in the catalytic pocket of b4Gal-T1, which is conserved among all vertebrate homologs, when mutated to Leu or Ile broadens the donor substrate specificity of the enzyme to 2substituants of galactose i.e., GalNAc or 2-keto-galactose or 2-azido-galactose. These mutants have been used for linking glycoconjugates via glycan chains (see Project # Z01 BC 010742). In invertebrates in the b4Gal-T homologs there is an Ile residue at the corresponding position of Tyr and they are b4GalNAc-T enzymes. We have shown that mutation of the Ile residue to Tyr in Drosophila b4GalNAc-T1 converts the enzyme to a b4Gal-T1 by reducing its N-acetylgalactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold. Mutation of this residue played an important role in the evolutionary divergence of invertebrate and vertebrate glycoconjugates (reported in the 2006 annual report). We have also shown that mutation of Arg228, a residue in the vicinity of Glu317, to lysine (R228K-Gal-T1) results in a 15-fold higher glucosyltransferase (Glc-T) activity, the transfer of glucose from UDP-Glc. This activity is further enhanced by LA to nearly 25% of the galactosyltransferase (Gal-T) activity of the wild type. This has been reported in our previous annual report.</p> <P><i><b>Few mutations in the catalytic domain of bovine alpha-1,3-galactosyltransferase (a3Gal-T) brodens the donor specificity:</i></b> We now show that (a3Gal-T) transfers galactose (Gal), and not N-acetylgalactosamine (GalNAc), from its UDP-derivative to an acceptor LacNAc (Gal-b1-4GlcNAc). The enzyme belongs to a3Gal/GalNAc family of glycosyltransferases to which human blood group transferases A and B belong. The blood group A transferase (a3GalNAc-T-A) and the B transferase (a3Gal-T-B), transfer GalNAc and Gal to the fucosylated LacNAc acceptor, Fuc-a1-2Gal-b1-4GlcNAc, respectively. The family shares common features, e. g., (1) they all use UDP-nucleotide as a sugar donor, (2) after the transfer reaction they retain the configuration at the anomeric C-atom of the donor sugar, Gal or GalNAc, and (3) their activity strictly depends on the presence of a divalent metal ion Mn<sup>2+</sup>. The catalytic domain of a3Gal-T exhibits very high sequence similarity with the corresponding region of the blood group A and B transferases. Whereas A and B transferases have identical amino acid sequence except for four amino acid residues, two of these residues, at positions 266 and 268, (Leu and Gly in A enzyme and Met and Ala in B enzyme) determine the sugar-donor specificity of the enzyme. In a3Gal-T at corresponding positions have His and Ala residues, respectively. Mutating these residues to Leu/Ser/Thr and Gly increases the GalNAc activity of the a3Gal-T to about 5-14% of the Gal-T activity. Additionally a mutation close to DVD motif of glutamine (Gln) to methionine (Met) improved a3Gal-T activity for GalNAc to 59%. Close examination of the structures of these enzymes reveal that the three dimensional crystal structures of a3Gal-T and the blood group A transferases (a3GalNAc-T-A) are quite similar except for the missing residues in a3GalNAc-T-A structure that correspond to residues 179-194 in a3Gal-T, which adopt an alpha-helix region (residues 179-187) and a hinge region (188-194). We hypothesize that the alpha-helix and the hinge regions of the a3Gal-T undergo a helix-loop transition during catalysis that allows the sugar nucleotide to enter the catalytic pocket. Thus, to increase the flexibility of the helix region, in addition of mutating His280-Ala281-Ala282 of a3Gal-T to Ser280-Gly281-Gly282, Q228 to Met228, we mutated Pro191 in the hinge region to Ala191 and S191. These mutations have enabled us to convert a3Gal-T to an a3GalNAc-T that transfers GalNAc to LacNAc with an activity that is comparable to 28-59% activity of the wild type towards Gal. These mutants also transfer modified galactose with a chemical handle e.g., 2-keto-galactose, to LacNAc residues on glycoproteins, making it possible to use these mutants, (1) for the detection of alterations in the glycosylation patterns in many pathological states, such as cancers and rheumatoid arthritis, and (2) in the glycoconjugation and assembly of nano-particles for the targeted drug delivery of bioactive-agents. </p